The human LINE-1 (L1Hs) element is a transposon that causes insertional mutagenesis upon transposition into sensitive genomic loci. Examples of both somatic and presumed germ-line insertional mutagenesis events have been reported and new examples are being discovered regularly. About 5% of the human genome is composed of the estimated 4000 full length and 100,000 truncated L1Hs elements that it contains.Expression of L1Hs is highly regulated and occurs mostly in cells of germ line origin. The sequences necessary and sufficient for the appropriate expression of L1Hs are found within the elements 5~ UTR. We have continued our investigation of a 140 bp fragment of the L1Hs 5~ UTR that we previously showed acts as an enhancer in NTera2D1 human teratocarcinoma cells. Partial deletions of this fragment results in partial decrease in activity and partial fragments contain enhancer activity when cloned into heterologous promoters suggesting that the intact fragment contains more than one enhancer sequence. Mutations of a motif within the fragment that we previously showed is footprinted by nuclear proteins from NTera2D1 cells in DNAse I protection experiments results in increased enhancer activity suggesting that this sequence may be acting as a transcriptional silencer. We are continuing to narrow down the regions of DNA that contain the enhancers by deletion analysis. A recent search of the Genbank revealed a strong similarity between a motif in the fragment and a previously unstudied region of the JC virus regulatory region. A related sequence in other genes is known to bind to members of the HMG family of DNA binding proteins. We are testing the hypothesis that an HMG protein family member is acting as a transcriptional regulating factor of L1Hs.During the past year we have continued our efforts to investigate the expression of L1Hs in M28 cells, a somatic cell hybrid line containing a human isochromosome 12p. This abnormal chromosome is a feature of both testicular germ cell tumors and the Pallister-Killian Syndrome. Our studies indicate that the similar but not identical regions of the L1Hs 5~ UTR govern expression of L1Hs in NTera2D1 and M28 cells. Experiments designed to clone the gene responsible for the enhanced expression of L1Hs in M28 cells are under way.